雷帕霉素通过诱导自噬对骨髓间充质干细胞的成骨作用。
Osteogenic effects of rapamycin on bone marrow mesenchymal stem cells via inducing autophagy.
机构信息
Fujian Key Laboratory of Oral Diseases & Fujian Provincial Engineering Research Center of Oral Biomaterial & Stomatological Key Lab of Fujian College and University, School and Hospital of Stomatology, Fujian Medical University, Fuzhou, China.
Institute of Stomatology & Research Center of Dental and Craniofacial Implants, School and Hospital of Stomatology, Fujian Medical University, Fuzhou, China.
出版信息
J Orthop Surg Res. 2023 Feb 22;18(1):129. doi: 10.1186/s13018-023-03616-9.
BACKGROUND
While autophagy is essential for stem cells' self-renewal and differentiation, its effect on bone marrow mesenchymal stem cells (BMSCs) remains unclear. This study aimed to investigate the interaction between autophagy and osteogenic differentiation using rapamycin (RAPA), a classical autophagy agonist with osteo-regulatory effects.
METHODS
Rat BMSC's autophagy was analyzed after osteoinduction (0, 7, 14, and 21 d) by western blotting, immunofluorescence, and real-time quantitative polymerase chain reaction (RT-qPCR). In addition, we evaluated osteogenic differentiation using alizarin red staining, alkaline phosphatase assays, and RT-qPCR/Western blotting quantification of bone sialoprotein, type 1 collagen, alkaline phosphatase, osteopontin, and Runt-related transcription factor 2 mRNA and protein levels.
RESULTS
The BMSC's basal autophagy level gradually decreased during osteogenic differentiation with a decrease in BECN1 level and the lipidated (LC3-II) to unlipidated (LC3-I) microtubule-associated protein 1 light chain 3 ratio and an increase in the expression of selective autophagic target p62. In contrast, it increased with increasing RAPA concentration. Furthermore, while 2 nM RAPA promoted BMSC osteogenic differentiation on days 7 and 14, 5 nM RAPA inhibited osteogenesis on days 14 and 21. Inhibition of autophagy by the inhibitor 3-methyladenine could impair RAPA's osteogenesis-enhancing effect on BMSCs.
CONCLUSIONS
The BMSC's basal autophagy level decreased over time during osteogenic differentiation. However, an appropriate RAPA concentration promoted BMSC osteogenic differentiation via autophagy activation.
背景
自噬对于干细胞的自我更新和分化至关重要，但它对骨髓间充质干细胞（BMSCs）的影响尚不清楚。本研究旨在使用具有骨调节作用的经典自噬激动剂雷帕霉素（RAPA）来研究自噬与成骨分化之间的相互作用。
方法
通过 Western blot、免疫荧光和实时定量聚合酶链反应（RT-qPCR）分析成骨诱导（0、7、14 和 21 天）后大鼠 BMSC 的自噬。此外，我们通过茜素红染色、碱性磷酸酶测定和 RT-qPCR/Western blot 定量骨涎蛋白、I 型胶原、碱性磷酸酶、骨桥蛋白和 runt 相关转录因子 2 mRNA 和蛋白水平来评估成骨分化。
结果
在成骨分化过程中，BMSC 的基础自噬水平逐渐降低，BECN1 水平降低，微管相关蛋白 1 轻链 3 （LC3）的脂质化（LC3-II）与非脂质化（LC3-I）的比率降低，选择性自噬靶标 p62 的表达增加。相比之下，随着 RAPA 浓度的增加，自噬水平也随之增加。此外，2 nM RAPA 在第 7 和 14 天促进 BMSC 成骨分化，而 5 nM RAPA 在第 14 和 21 天抑制成骨。自噬抑制剂 3-甲基腺嘌呤可抑制自噬，从而削弱 RAPA 对 BMSCs 成骨增强作用。
结论
在成骨分化过程中，BMSC 的基础自噬水平随时间逐渐降低。然而，适当浓度的 RAPA 通过激活自噬促进 BMSC 成骨分化。
Zotero 插件